Scaffold supports results from MaxQuant, a free search engine created and supported by the Max Planck Institute. MaxQuant users have access to numerous quantitative options including reporter ion, SILAC or precursor intensity techniques. The following document gives instructions for configuring MaxQuant in order to load data into Scaffold. Note, when loading MaxQuant data into Scaffold users should navigate to and select the entire combined directory using the file browser. If specific errors arise please contact Proteome Software Technical Support by phone Monday through Friday 8:00 AM to 5:00 PM Pacific Time at 1-800-944-6027 or by email at firstname.lastname@example.org.
Please see the Scaffold File Compatibility Matrix for the most up to date information on search engines currently supported in Scaffold.
General MaxQuant Processing Steps
The following steps give a few details on configuring a MaxQuant search.
- Raw files are loaded using the Raw files tab, files can either be loaded individually or by selecting a directory.
- If the files are not fractions select the “No Fractions” button. This will allow for files to be loaded into Scaffold individually from the combined directory
- Panes in the Group-specific parameters tab allow for the setting of modifications, digestion enzyme, and instrument settings.
- The Global-parameters tab contains panes to define the FASTA file, identification parameters and additional instrument settings.
These settings can be used for most data processing in MaxQuant and the Quantitation type can be changed as needed based on instructions below. Note, MaxQuant by default writes output files to the same folder as the raw data and will overwrite raw data files if the same raw file is researched. It is thus recommended to move the output files to a new folder before raw data is reprocessed.
Suggested FDR settings
In most instances, MaxQuant’s supporting documentation recommends running the program with default settings. However, in order for Scaffold to have access to enough data to properly build probability distributions, Proteome Software suggests users increase the peptide and protein FDR values. This can be accomplished by setting the “PSM FDR and “Protein FDR” entries found in the Global Parameter’s Identification tab to 1.0 (note the default is 0.01 or 1%).
Scaffold 4.8.1 introduced Pre-Filtered Mode which allows users to bypass Scaffold’s built-in probability model and filter your data using the search engine instead. If you would like to use this option you should not set the FDR value to 1.0 as described in the section above but set the value to an acceptable FDR cutoff. If you use Pre-Filtered Mode you will not be able to filter your data in Scaffold using the Protein and Peptide dropdown menus.
The Pre-Filtered option is found in the Scoring System section of the Load and Analyze Data step of Scaffold’s Load Data Wizard
Selecting Results in Scaffold
Maxquant allows users to process multiple raw files at once. Scaffold allows for these files to be loaded all into one biosample or broken out into multiple biosamples as is appropriate for the data. A dialog box with appear when users point to a Maxquant combined directory with multiple raw files. To proceed, click on one or more of the raw files in the dialog to highlight them. Then click OK. If you do not select one of the files in the dialog you will get an error indicating the Scaffold cannot load any MS/MS data.
Precursor Intensity Quantitation Settings
Scaffold and the Q+ quantitation modules support precursor ion intensity quantitation results from MaxQuant. To set up precursor ion quantitation the Dropdown for Label-free quantification in the Group-specific parameters tab should be changed from None to LFQ; the “Type” dropdown found in the Group-specific parameters tab should be left on Standard. The output files should now contain precursor ion intensities.
The LFQ setting enables precursor intensity quantitation
As precursor intensity quantitative results are not multiplexed into a single files as with SILAC or reporter ion quantitation Scaffold requires MS sample files to be loaded into multiple Biosamples. In MaxQuant 1.5 and above this can be accomplished in two ways:
- Single raw files can be analyzed separately and the individual files can be loaded into separate Biosamples
- Multiple raw files can be searched at once if the “No fractions” option is selected. This will create an individual MS sample file for each raw file. When the combined directory is selected in Scaffold an additional dialog box will appear allowing individual files to be chosen. Simply select the same combined directory and load the proper files into each Biosample.
Reporter Ion Quantitation Settings
For samples with isobaric tags, iTRAQ or TMT, MaxQuant gives users the option to use either MS2 or MS3 spectra. The “Type” dropdown found in the Group-specific parameters should be set to either MS2 or MS3 depending on how the experiment was set up. When the type of the experiment is defined as available. Select the number of tags that correspond to your experimental design.
SILAC and Dimethyl Quantitation Settings
Finally, MaxQuant produces SILAC and dimethyl quantitative results that are compatible with Scaffold. The “Type” should be set to Standard and instead the multiplicity should be changed to match the number of channels in the multiplex (set this to 2 for SILAC 2-plex or 3 for SILAC 3-plex). Once the multiplicity is set the program should prompt for the modifications used for the light, medium and heavy channels. Once the run completes the output can be loaded into Scaffold Q+S and the experiment should contain the quantitative information.
Importing Custom Modifications from MaxQuant
Scaffold comes bundled with a generic copy of the modifications.xml file found in MaxQuant and is used to properly name modifications appearing in search results. This file stores all of MaxQuant’s modification information and is edited when users add custom modifications. Thus, this file will have to be manually updated in Scaffold to reflect the changes. Follow the following steps to update Scaffold’s modifications.xml file:
- The modifications file can be found in the MaxQuant installation directory: \MaxQuant_<version_number>\MaxQuant\bin\conf\modifications.xml.
- Copy this file
- Navigate to the Scaffold installation directory found here: C:\Program Files\Scaffold 4.8.3\parameters\maxquant
- Rename the modification.xml file modification.xml-back
- Paste the file from MaxQuant into this folder. Once you restart Scaffold the new file should be used for naming custom modifications.
Files Read By Scaffold in Combined Directory
There are numerous files written to the combined directory by default. The following lists what files are actually read by Scaffold when data is loaded.
MaxQuant version 1.3, the files that Scaffold reads are:
All APL files:
For MaxQuant versions 1.4-1.6 (note the andromeda directory inside combined)
-All APL files: